Arrangement and Method for the Electrochemical Analysis of Liquid Samples by Means of Lateral Flow Assays

ABSTRACT

The invention relates to an arrangement ( 1 ) and to a method for the electrical detection of liquid samples ( 5 ) by means of lateral flow assays. The lateral flow assay comprises a membrane ( 4 ) that is arranged on a front side of a first carrier ( 2 ). The first carrier ( 2 ) is electrically insulating. On the front side of the first carrier ( 2 ) between the carrier ( 2 ) and the membrane ( 4 ), electrically conductive electrodes ( 3 ) are arranged in direct contact with the membrane ( 4 ).

The present invention relates to an arrangement and a method for theelectrical detection of liquid samples by means of lateral flow assays,wherein the lateral flow assay comprises a membrane arranged on a frontside of a first carrier. The first carrier is embodied in anelectrically insulating fashion and electrically conductive electrodesare arranged on this carrier.

Lateral flow assays are in widespread use in in-vitro diagnostics (IVD).They are simple in terms of handling and very cost-effective.Disadvantages of lateral flow assays include a low sensitivity, a lowmultiplexity and a poor quantifiability of the results.

A good quantifiability can be achieved by means of optical, magnetic andelectrical methods, but heretofore with very low multiplexity, i.e.simultaneous measurement at a plurality of spatially separatemeasurement points.

U.S. Pat. No. 6,896,778 discloses an arrangement in which, for a goodmultiplexity, gold electrodes are arranged above cutouts of anelectrically insulating carrier as an array, and the cutouts are filledwith membranes composed of a polymer/microfiber matrix material, saidmembranes being spatially separated from one another. The membranes areion-selective, and not suitable e.g. for immunosensors in immunoassays.

With the use of capture antibodies in immunoassays, these have to beimmobilized directly on the gold electrodes or sensors. In anarrangement analogous to the laminated arrangement of a carriercomprising insulator layers and gold electrodes that is described inU.S. Pat. No. 6,896,778, wherein the gold electrodes are arranged abovecutouts in the insulator layers in array form, a small cavity in eachcase arises above the gold electrodes through the surrounding insulatorlayer. When liquid is applied directly or via a lateral flow paper as amembrane above the arrangement, air bubbles arise in the region of thecavities and, during a measurement, lead to a failure of the respectiveelectrodes with air inclusions.

Therefore, it is an object of the present invention to specify anarrangement and a method for the electrical detection of liquid sampleswhich enable a good multiplexity in conjunction with very goodsensitivity and quantifiability. In this case, good multiplexity shouldbe understood to mean a multiplexity e.g. in the range of 3- to 10-plex(sensors). In particular, it is an object to specify an arrangement anda method which enable a reliable measurement, in particular withoutdisturbing air inclusions above the electrodes.

The object specified is achieved with regard to the arrangement for theelectrical detection of liquid samples by means of lateral flow assayswith the features of claim 1 and with regard to the method for theelectrical detection of liquid samples by means of the above-describedarrangement with the features of claim 11.

Advantageous configurations of the arrangement according to theinvention for the electrical detection of liquid samples by means oflateral flow assays and of the method for the electrical detection ofliquid samples by means of the above-described arrangement are evidentfrom the respectively assigned dependent claims. In this case, thefeatures of the main claim can be combined with features of thedependent claims and features of the dependent claims can be combinedamong one another.

The arrangement according to the invention for the electrical detectionof liquid samples by means of lateral flow assays comprises a membranearranged on a front side of a first carrier. The first carrier isembodied in an electrically insulating fashion and electricallyconductive electrodes are formed on the first carrier. The electrodes onthe front side of the first carrier are arranged between the carrier andthe membrane, in direct contact with the membrane.

By virtue of the arrangement of the electrodes on the front side of thefirst carrier, i.e. on the side on which the membrane is also arranged,the electrodes can form a direct contact with the membrane. Theformation of a hollow space or of a cavity, such as is present e.g. inthe prior art described above, is thereby prevented. The contact area ismaximized with the membrane arranged flat on the electrode and, when theliquid sample to be analyzed is applied to the membrane, the electrodesare in direct contact with the liquid sample. Air bubbles or airinclusions which can impede or completely prevent a measurement areprevented by the direct contact of electrodes and membrane, and thusalso the direct contact of electrodes and the liquid sample. As aresult, a reliable measurement of the sample is made possible, includingin the case of multiplex measurement (with a plurality of sensorssimultaneously), with very good sensitivity and quantifiability of thesample.

The membrane can be embodied as a closed layer via which the electrodes,in particular all the electrodes, are connected to one another. Thisenables a lateral liquid transport (lateral flow) completely via themembrane in particular via all the electrodes.

The membrane can comprise or be a lateral flow paper, in particularcomposed of nitrocellulose. Lateral flow paper has a high porosity andabsorbs the liquid sample well and transports it well to the electrodesand leads there to a good wetting of the electrodes with the sampleliquid to be examined. A good electrical contact via membrane saturatedwith liquid sample between electrodes is thus made possible.Nitrocellulose is cost-effective and, used as a membrane, has theproperties described above.

The electrodes can be metal electrodes, in particular composed of gold.Electrodes composed of metal are generally relatively stable, and goldelectrodes, in particular, can be used well electrochemically since theycan lead to temporally stable measurement signals and chemically aresubstantially inert.

The electrodes can be electrically contact-connected on the front sideof the carrier, in particular in an edge region in which the membrane isnot arranged. This affords advantages in particular if the rear sidecannot readily be reached for electrical contacts e.g. as a result ofencapsulation.

However, the carrier can also have in each case in the region of arespective electrode an opening passing through its thickness, from thefront side to the rear side, through which opening the electrode iselectrically contact-connected conductively from the front side to therear side of the carrier. This makes it possible to prevent electricalshort circuits between electrode contacts e.g. upon contact with sampleliquid on the front side of the carrier.

The carrier can comprise a plurality of insulating layers, in particularlayers composed of polymers and/or layers which are connected to oneanother by lamination. Laminated carriers composed of polymers areprinted circuit boards, for example which can be producedcost-effectively.

The membrane can be arranged in a sandwich-like fashion firstly betweenthe front side of the first carrier in direct contact with theelectrically conductive electrodes (working electrodes) of the firstcarrier and secondly a rear side of a second carrier in direct contactwith at least one electrode (counterelectrode), in particular exactlyone electrode on the rear side of the second carrier. This arrangementenables a compact construction and short paths via the membrane betweenelectrodes in order to establish a voltage between the workingelectrodes and the counterelectrode.

Each electrode on the front side of the first carrier can be in eachcase electrically connected to the at least one electrode on the rearside of the second carrier, in particular via an electrical measuringinstrument or measuring device for measuring current and/or voltageand/or capacitance. The electrodes can be arranged on the front side ofthe first carrier in a series in tandem or in array form. As a result,an electrochemical measurement is made possible and a spatially resolvedelectrochemical measurement of the liquid sample in the membrane can becarried out analogously to an optical measurement in chromatography.

The method according to the invention for the electrical detection ofliquid samples is effected by means of an arrangement described above.The liquid sample is applied to the membrane and is moved by means ofcapillary forces, in particular, via the membrane to the electrodes. Themembrane interconnects the electrodes, in particular electrochemicallyif the membrane is filled with liquid. In particular, exactly onemembrane interconnects in particular all the electrodes. As a result, agood conductivity is ensured in the case of conductive liquid betweenthe electrodes via the one membrane.

The arrangement brings about according to the lateral flow method aspatial and/or temporal separation of substances in the liquid sampleanalogously to chromatography or with different capture moleculesimmobilized at different locations. The spatial and/or temporalseparation can be measured electrochemically by means of the electrodesin the form of current and/or voltage and/or charge changes.

In the case of the construction described, the membrane can be in directcontact with the electrodes. In each case the contact area of eachelectrode with the membrane can thus be completely wetted with theliquid sample, in particular without air inclusions above the electrode.This ensures a good electrochemical measurement by means of theelectrode, which would be prevented or at least impeded e.g. by airbubbles directly above the electrode.

The liquid sample can be a biochemical sample, in particular a bodyfluid. In this regard, e.g. urine, blood, or the information thereof canbe examined.

The advantages associated with the method for the electrical detectionof liquid samples by means of the arrangement described above areanalogous to the advantages described above with regard to thearrangement for the electrical detection of liquid samples by means oflateral flow assays.

Preferred embodiments of the invention with advantageous developments inaccordance with the features of the dependent claims are explained ingreater detail below with reference to the figures, but without beingrestricted thereto.

In the figures:

FIG. 1 illustrates a schematic sectional illustration through anarrangement 1 for the electrical detection of liquid samples 5comprising a laminated electrode 3 and an ion-selective membrane 4arranged thereabove according to the prior art, and

FIG. 2 illustrates a schematic sectional illustration through anarrangement 1 according to the invention for the electrical detection ofliquid samples 5 comprising a laminated electrode 3 for lateral flowassays, wherein the electrode 3 on the front side of a carrier 2 isarranged between carrier 2 and membrane 4, and

FIG. 3 illustrates a schematic sectional illustration through anarrangement 1 analogous to that in FIG. 2, comprising a plurality ofelectrodes 3 arranged in series in tandem and without electrical contact6 from the rear side of the arrangement 1, and

FIG. 4 illustrates a schematic illustration in a plan view of thearrangement 1 shown in FIG. 3 with a strip of lateral flow paper 4placed onto the electrodes 3 in such a way that a lateral region remainsfree for an electrical contact 6, and

FIG. 5 illustrates a schematic sectional illustration through anarrangement 1 analogous to that shown in FIG. 3, comprising a secondelectrode carrier 8 with counterelectrode 9 arranged on the membrane 4and measuring devices 10 in each case connected to a working electrode 3of the first carrier 2 and the counterelectrode 9 of the second carrier8.

The arrangement 1 for the electrical detection of liquid samples 5according to the prior art as shown in FIG. 1 has a laminated electrode3 of an electrode array. The arrangement 1 is shown schematically insectional illustration. The electrode 3 is arranged below a firstcarrier 2, which is constructed e.g. from laminated polymer layersanalogously to a printed circuit board, wherein the electrode 3 islaminated e.g. as a gold layer onto the carrier 2. However, theelectrode can also e.g. be adhesively bonded or appliedelectrolytically.

A cutout passing through the carrier 2 is introduced in the carrier 2,above the electrode 3. Said cutout can be embodied e.g. in the form of adrilled hole or milled hole e.g. in a circular fashion. Arranged in thecutout, in contact with the electrode 3, is a membrane 4 as anion-selective layer, which completely covers the free area of theelectrode 3 in the cutout.

The electrode 3 is electrically contact-connected via an electricalcontact 6 from the rear side, i.e. from the side of the electrode 3which is opposite relative to the membrane 4 and is not covered by themembrane 4. A liquid sample 5 is guided via the membrane 4 and that sideof the carrier 2 on which no electrodes 3 are arranged and which isopposite relative to the side with the electrodes on the carrier 2, saidliquid sample being electrochemically in contact with the electrode 3via the membrane 4. That means that ions can move through the membrane 4from the liquid samples 5 to the electrode 3.

A counterelectrode, not shown for the sake of simplicity, is inelectrical contact with the liquid sample 5. Between thecounterelectrode and the electrode 3, which functions as a workingelectrode, a current, voltage and/or charge change at the electrode 3can be measured by means of a measuring device 10 (not shown). Themeasurement is carried out depending on the liquid sample 5 and servesfor analyzing the sample 5. The measurement can be carried out dependingon the sample flow rate and/or time and provides information about thecomposition or chemical/biochemical constituents of the sample 5. By wayof example, blood, urine or other body fluids can serve as samples 5.However, other liquids through to gases can also be examined.

The arrangement 1 shown in FIG. 1 can be used in a sensor array or someother arrangement of sensors, e.g. in series. The electrodes 3constitute the sensors. For a reliable measurement it is important thatno liquid 5 passes to that side of the first carrier 2 with theelectrodes 3 on which the electrical contacts 6 are secured.

One disadvantage of the construction described above is theion-selective membrane 4. The membrane 4 is not suitable forimmunosensors, for example. The complicated construction is costly toproduce and difficult to handle.

FIG. 2 illustrates a schematic sectional illustration through anarrangement 1 according to the invention for the electrical detection ofliquid samples 5. An electrode 3 is arranged on a first, laminatedcarrier 2, analogously to the arrangement 1 described above under theprior art. The membrane 4 is arranged on the front side of the electrode3, which according to the invention is opposite relative to the side ofthe electrode 3 in contact with the carrier 2. The membrane 4 is lateralflow paper, for example, which lies flat on the electrode.

In the exemplary embodiment illustrated in FIG. 2, the electrode 3 iselectrically contact-connected via an electrical contact 6 on the rearside of the electrode 3. In the laminated carrier 2, in the region ofthe electrode 3 on the rear side thereof there is a cutout through whichthe electrical contact 6 projects as far as the electrode 3. Asdescribed below, the electrode 3 can also be contact-connected from thefront side.

FIG. 3 and FIG. 4 show an exemplary embodiment of the arrangement 1according to the invention in which an electrical contact 6 of theelectrodes 3 can be effected laterally on the front side. FIG. 3illustrates a schematic sectional illustration through an arrangement 1analogous to that shown in FIG. 2, but with a plurality of theelectrodes 3 shown in FIG. 2 arranged in series in tandem. Theelectrodes 3 can also be arranged as an array, e.g. in a matrix-typefashion on the first carrier 2.

FIG. 4 illustrates a plan view of the arrangement 1 shown in FIG. 3 withelectrodes 3 in a series 7. A membrane 4, e.g. composed of lateral flowpaper, is arranged in strip form on the electrodes 3 in such a way thata lateral region of the electrodes 3 remains free for an electricalcontact. In the lateral region, electrical contacts 6 can be fitted ineach case to each electrode 3, this not being illustrated in the figuresfor the sake of simplicity.

FIG. 5 shows a construction for an electrical measurement with thearrangement 1 according to the invention as shown in FIGS. 3 and 4, in aschematic sectional illustration. A second carrier 8 with an electrode 9is arranged on the membrane 4, wherein the membrane 4 bears in directcontact flat against the electrode 9. The electrode 9 serves as acounterelectrode. Below the membrane 4, a series of electrodes 3 servingas working electrodes is arranged in direct contact with the membrane 4.The electrodes 3 are arranged on a carrier 2, as already shown in FIGS.2 to 4. Consequently, the membrane 4 lies in a sandwich-like fashionbetween the electrodes 3 on the first carrier 2 and the electrode 9applied in a planar fashion on the second carrier 8.

As shown schematically in FIG. 5, for a measurement the electrode 9 canbe electrically connected to each electrode 3 in each case via anelectrical measuring instrument or a measuring device 10. In this case,the electrodes 3, 9 can be electrically contact-connected from the sidewith the carrier 2, 8 through openings in the carrier 2, 8 as shown inFIG. 2, or, as described for FIG. 4, from that side of the electrodes 3,9 which is in direct contact with the membrane 4, but in a region of theelectrodes 3, 7 in which the membrane 4 is not arranged.

If a liquid sample 5, e.g. blood or urine, is applied to the membrane 4,e.g. lateral flow paper comprised of nitrocellulose in strip form or insome other form, the sample 5 is moved into and through the membrane 4e.g. through the porous structure of the membrane 4. The membrane 4 isthus “filled” with the sample 5. As a result of the electricalconductivity of the sample 5, the electrodes 3, 9 in direct contact withthe membrane 4 are electrically or electrochemically connected to oneanother, and a closed electric circuit is provided in each case via themeasuring device 10 between the respective electrode 3 as workingelectrode and the electrode 9 as counterelectrode. With electrochemicalmeasurements, in the case of an electrode array or electrodes 3 arrangedin series, the composition of the liquid sample can be examined in aspatially and temporally resolved manner. In this regard, at theindividual electrodes 3 in a location-related fashion, i.e. at thelocation of the electrode 3, electrochemical measurements via current,voltage and/or capacitance measurements can provide information aboutthe sample 5 situated at the location.

Analogously to chromatographic examinations, the sample composition canbe analyzed or capture molecules can be immobilized on the respectiveelectrode 3, e.g. different capture molecules on different electrodes 3,and enable the detection of individual substances in the sample 5. Thecapture molecules can also be immobilized in a spatially distributedmanner in the membrane 4 in the region above the electrode 3. As aresult, the arrangement 1 according to the invention can be used inimmunoassays.

The electrodes 3, 9 can be used as illustrated in the figures, see FIG.5 in particular, in a measurement set-up comprising working electrode 3and counterelectrode 9. In addition, at least one reference electrodeRE, not illustrated in the figures for the sake of simplicity, can alsobe used. Metal layers, in particular composed of gold or platinum, canbe used as electrodes or silver/silver chloride layers or electrodes canbe used e.g. as reference electrode. Other measurement set-ups that arecustomary in electrochemistry are also possible.

The exemplary embodiments described above can be used in combination.The exemplary embodiments can also be combined with exemplaryembodiments known from the prior art. In this regard, besides lateralflow paper e.g. composed of nitrocellulose, membranes 4 composed ofpolyvinylidene fluoride, electrostatically treated nylon orpolyethersulfone can be used as membranes 4. Electrodes 3, 9 can beapplied in planar fashion on the carrier 2, 8 or in a spatiallystructured fashion, in series in tandem, in array form in a n×m matrixcomprising n lines and m columns, or else can be provided with differentheight profiles.

1. An arrangement (1) for the electrical detection of liquid samples (5)by means of lateral flow assays, wherein the lateral flow assaycomprises a membrane (4) arranged on a front side of a first carrier(2), and wherein the first carrier (2) is embodied in an electricallyinsulating fashion and electrically conductive electrodes (3) arearranged on the first carrier (2), characterized in that the electrodes(3) on the front side of the first carrier (2) are arranged between thecarrier (2) and the membrane (4), in direct contact with the membrane(4).
 2. The arrangement (1) as claimed in claim 1, characterized in thatthe membrane (4) is embodied as a closed layer via which the electrodes(3, 7), in particular all the electrodes (3, 7) are connected to oneanother.
 3. The arrangement (1) as claimed in either of the precedingclaims, characterized in that the membrane (4) comprises a lateral flowpaper, in particular composed of nitrocellulose.
 4. The arrangement (1)as claimed in any of the preceding claims, characterized in that theelectrodes (3, 7) are metal electrodes, in particular composed of gold.5. The arrangement (1) as claimed in any of the preceding claims,characterized in that the electrodes (3, 7) are electricallycontact-connected on the front side of the carrier (2, 8), in particularin an edge region in which the membrane (4) is not arranged.
 6. Thearrangement (1) as claimed in any of claims 1 to 4, characterized inthat the carrier (2, 8) has in each case in the region of a respectiveelectrode (3, 7) an opening passing through its thickness, from thefront side to the rear side, through which opening the electrode (3, 7)is electrically contact-connected conductively from the front side tothe rear side of the carrier (2, 8).
 7. The arrangement (1) as claimedin any of the preceding claims, characterized in that the carrier (2, 8)comprises a plurality of insulating layers, in particular layerscomposed of polymers and/or layers which are connected to one another bylamination.
 8. The arrangement (1) as claimed in any of the precedingclaims, characterized in that the membrane (4) is arranged in asandwich-like fashion between the front side of the first carrier (2) indirect contact with the electrically conductive electrodes (3) of thefirst carrier (2) and a rear side of a second carrier (8) in directcontact with at least one electrode (7), in particular exactly oneelectrode (7) on the rear side of the second carrier (8).
 9. Thearrangement (1) as claimed in claim 8, characterized in that eachelectrode (3) on the front side of the first carrier (2) is in each caseelectrically connected to the at least one electrode (7) on the rearside of the second carrier (8), in particular via an electricalmeasuring device (10) for measuring current and/or voltage and/orcapacitance.
 10. The arrangement (1) as claimed in any of the precedingclaims, characterized in that the electrodes (3) are arranged on thefront side of the first carrier (2) in a series (7) in tandem or inarray form.
 11. A method for the electrical detection of liquid samples(5) by means of an arrangement (1) as claimed in any of the precedingclaims, characterized in that the liquid sample (5) is applied to themembrane and is moved by means of capillary forces, in particular, overthe membrane to the electrodes (3, 7), characterized in that themembrane (4), in particular exactly one membrane (4), interconnects theelectrodes (3, 7), in particular all the electrodes (3, 7).
 12. Themethod as claimed in claim 11, characterized in that the arrangement (1)brings about, according to the lateral flow method, a spatial and/ortemporal separation of substances in the liquid sample (5) which ismeasured by means of the electrodes (3, 7) in the form of current and/orvoltage and/or charge changes.
 13. The method as claimed in either ofclaims 11 and 12, characterized in that the membrane (4) is in directcontact with the electrodes (3, 7) and in each case the contact area ofeach electrode (3, 7) with the membrane (4) is completely wetted withthe liquid sample (5), in particular without air inclusions.
 14. Themethod as claimed in any of claims 11 to 13, characterized in that theliquid sample (5) is a biochemical sample (5), in particular a bodyfluid.